Chemical compounds and methods for preparing the same



United States Patent 3,462,487 CHEMICAL COMPOUNDS AND METHODS FOR PREPARING THE SAME Roland Walter Kinney, Trenton, Saul Lewis Neidleman,

Highland Park, and Frank Lee Weisenborn and John 5 Samuel Paul Schwarz, Somerset, N.J., assignors, by mesne assignments, to E. R. Squibb & Sons, Inc., New York, N.Y., a corporation of Delaware No Drawing. Filed June 9, 1964, Ser. No. 373,850

Int. Cl. C07c 103/19 US. Cl. 260-559 10 7 Claims ABSTRACT OF THE DISCLOSURE Ttetracycline derivatives are prepared by subjecting tetracycline, 6-demethyltetracycline or S-hydroxytetracycline under aerobic conditions to the action of peroxidase in the presence of dihydroxyfumaric acid.

This invention relates to new chemical compounds and new processes for preparing chemical compounds, and, more particularly, to new tetracycline and secotetracycline derivatives and new processes for preparing such derivatives and other known tetracycline derivatives.

The new compounds of this invention include: (a) tetracycline derivatives of the Formula 1:

wherein the Rs are the same and are each hydrogen or acyl, and R is hydrogen or methyl; (b) lactone derivatives of secotetracyclines of the Formula II:

. wherein R" is hydrogen or a hydroxy group, and R'lS as hereinbefore defined; and (c) 4-dedimethylamino-4- keto-4a,l2a-dehydro-l2,12a secotetracycline-lZ-oic acid 6,12-lactone of the formula III:

III

5 i o n, H o H o The new compounds of this invention of Formula I include: 4-hydroxy 4 dedimethylaminotetracycline, 4-hydroxy-4-dedimethylamino-6-demethyltetracycline, and the 4,12a-diesters thereof, especially the diesters thereof with hydrocarbon carboxylic acids of less than twelve carbon atoms, as exemplified by the lower alkanoic acids (e.g., acetic, propionic, butyric and enanthic acid), the monocyclic aryl (lower alkanoic) acids (e.g., phenylacetic acid and fi-phenylpropionic acid), the monocyclic aryl carboxylic acids (e.g., benzoic acid and p-toluic acid), the lower alkenoic acids, the lower alkynoic acids, the cycloalkanecarboxylic acids and the cycloalkene carboxylic acids.

The new compounds of this invention of Formula II include: 4-dedimethylamino-12,1Za-secotetracycline-l2-oic acid 6,12-lactone, 4-dedimethylamino-5-hydroxy-12,12asecotetracycline-lZ-oic acid 6,12-lactone and 4-dedirnethylamino-6-demethyl-12,12a-secotetracycline-l2-oic acid 6, 12-lactone.

Those new compounds of this invention of the Formula I are therapeutically active compounds which possess broad spectrum antibacterial activity against many grampositive and gram-negative bacteria. Thus, the compounds of this invention can be administered perorally in the same manner as tetracycline in the treatment of bacterial diseases which respond to tetracycline treatment. In addition, the compounds of this invention display a high degree of activity against tetracycline-resistant microorganisms such as certain strains of Staphylococcus aureus, and hence are compounds of choice in treatment of diseases caused by such microorganisms.

Those new compounds of this invention of the Formula II and 4-dedimethylamino-4-keto-4a,l2a-dehydro-12,1221- secotetracycline-lZ-oic acid 6,12-lactone possess strong ultraviolet absorption properties, and so are ideally suited for use in sunburn preventive preparations, such as creams, lotions or oils, which are intended for topical application to filter out the ultraviolet radiation of natural sunlight. In addition, the new compound of this invention, 4 dedimethylamino-4-keto-4a,l2a-dehydro-l2,l2asecotetracycline-lZ-oic acid 6,12-lactone, is a strongly colored compound when viewed microscopically, possessing a dark, mustard-yellow color, and can therefore be used as a pigment for incorporation, together with a suitable vehicle such as linseed oil, for example, into artists paints and colors.

The new compounds of this invention are prepared by the process of this invention which comprises subjecting tetracycline, S-hydroxytetracycline or 6-demetl1yltetracycline under aerobic conditions to the action of the enzyme peroxidase in the presence of dihydroxyfumaric acid. The nature of the products formed will depend on the conditions under which the reaction is conducted, as more fully described hereinafter.

As sources of the peroxidase enzyme, plant cells and saps, animal tissues (such as liver), body fluids (such as saliva), leucocytes (myeloperoxidase), milk (lactoperoxidase) and many microorganisms may be used. The preferred sources of peroxidase for the purpose of this invention are horseradish and the microorganism, Myro thecium verrucaria. The peroxidase obtained from horseradish can be supplied merely by pressing horseradish and using the juice obtained or a purified preparation of horseradish peroxidase may be used. The peroxidase from M. verrucaria can be obtained by culturing the microorganism on a suitable nutrient medium, recovering the mycelium formed and treating the mycelium to recover purified peroxidase.

In addition to the peroxidase, dihydroxyfumaric acid is also added to the reaction mixture. Although substantially any concentration of this compound may be used, preferably the dihydroxyfumaric acid is present in a molar ratio of about 68 to 1 to about 1030 to 1 (optimally 3 about 480 to 1 to about 680 to 1) based on the weight of the tetracycline antibiotic. The reaction is preferably conducted at a pH in the range of about 3 to about 8 (optimally about 4.0 to about 6.0 and most advantageously at a pH of about 4.5). To assure that the pH of the reaction methylamino-6-demethyl-9-hydroxytetracycline, 4 dedimethylamino-6-demethyl-l2,12a secotetracycline-lZ-oic acid 6,12-lactone, and 4-dedimethylarnino-6-demethyl-4- hydroxytetracycline.

The 4,12a-esters of Formula I are prepared by reactmixture is maintained in this range, a buffering agent ing the 4-hydroxy-4-dedimethylamino derivatives of the which buffers in the desired pH range is preferably also respective tetracyclines with an acylating agent, such as added to the reaction medium. Suitable bufiiers include the acid anhydrides of the hydrocarbon carboxylic acids Mcllvaines buffer, potassium citrate buffer, potassium of less than twelve carbon atoms, as named hereinbefore, acetate buffer, potassium phosphate buffer and potassium 10 in a nonaqueous solution. After a suitable reaction period, formate buffer. the excess acid anhydride is destroyed with water in the The reaction is carried out in an aqueous medium under usual way, the solid esters are filtered off, washed well aerobic conditions, normally at a temperature in the with water, and dried. range of about C. to about 95 C. (optimally about 4-dedimethylamino-4-keto-4a,12a-dehydro-12,IZa-seco- 22 C. to about 37 C.). The components of the medium, 15 tetracycline-l2-oic acid 6,12-lactone and 4-hydroxy-4-denamely, the tetracycline, S-hydroxytetracycline or 6-dedimethylaminotetracycline can also be prepared nonenzymethyltetracycline, buffering agent, peroxidase and dimatically from tetracycline by treatment with a peracid, hydroxyfumaric acid (preferably after adjustment of pH such as m-chloroperbenzoic acid, to form the former and to the desired pH of the reaction medium, as by treatby treatment with hydrogen peroxide to form the latter. ment with a base, such as potassium hydroxide) are The following examples illustrate the invention (all merely mixed with water and the resultant mixture agitated temperatures being in Centigrade): or shaken to assure adequate aeration for about 10 to EXAMPLE 1 about 240 minutes (optimally about 30 minutes to about i 120 minutes) A reaction mixture of the following composition is Although the peroxidase acts merely as a catalyst and 25 Prepaffid! hence can be present in any proportion, to assure maxi- Volume Final mum conversion of the starting tetracycline to the de- (1111.) Component concentration sired final Products, the Peroxidase is Preset m a Welght 1.0 McIlvaine's butler, pH 4.5 (Handbook of ratio of about 0.1 to about 1.0 (optimally about 1.0) Chemistry and Physics, 35th ed. Qhem.

Rubber Publ. 00., Cleveland, Ohio, based on LhC tetracycline reactant. I page 1617),

A mixture of products is formed during the reaction Tetracycline hydrochloride 500 ug/ L which can be separated chromatographically as more gfiggg fi fully described in the examples following. Among these Distilled products, the following are formed if tetracycline is ern- $,;fi gxggfifiggifigggt? m pH ployed as the tetracycline reactant: the known compounds 4'dcdimethylaminotetracycline, 5a,6-anhydfotetrcychne, The reaction is initiated by the final addition of the enzyme. 5a,6-anhydro-4-dedimethylaminotetracyline, 4-ded1methyl- Th i ture i incubated in a glass tube, 25 x 100 mm., amino'9'hydroxytetracycllne, and the q p l at 25 on a rotary shaker with a 2-inch displacement at 4-dedimethylaminofl2,lza-sPcotetfacycllne-lZ-OIC field 6, 40 280 cycles/minute. At the end of 60 minutes, the reaction 124203006, 4-dfidimethylamlfio-4-hydfoxytetl'acyclme, and products are extracted from the aqueous solution into 4- y m hy r i 1.0 ml. of ethyl acetate. The extract is analyzed by paper tetracycline-12-oic acid 6,12-lactone. If S-hydroxytetrachromatography, Samples of 20 1. are spotted o Wh cycline is employed as the tetracycline reactant, the folman N 1 paper b ff d at H i h (105 M m lowing Compounds are formed: the known compound, 45 sium citrate and hydrated by dipping into an aqueous dedimethylamino-5-hydroxytetracycline and the new coml ti of 80% (v./v acetone and air-drying to evapo- Poufld, y y y- 3f' rate the acetone. The chromatograms are developed dec1ine-12-oic acid 6,12-lactone. If -demethyltetracyclme 1s scendingly with hexanezethyl acetate, 1:1 (v./v.) at room p y as th$ tetracycline madam, the fOHOWlIlg temperature. After development the dried chromatograms pounds are formed: 4-dedimethylamino-6-demethyltetraare examined visually under an ultraviolet lamp (Mincycline, 5a,6-anhydro-G-demethyltetracycline, 5a,6-anhyeralite Model SL, maximum emission at 254 m to dedro-4-dedimethylamino-6-demethyltetracycline, 4 deditect absorbing and fluorescing compounds. Spots are out- TABLE Inhibitory Fluorescence Fluorescence Product Rf activity before N113 after NHa Identity A 0.78 Orange Green 4-dedimethylaminotetracycline.

B 0.68 .do Orange 4-dedimethylamino- 12,12asecotetraeycline- 1Zoie acid 6,12-laetono.

C 0.68 Blue-green..-- Blue-grooml D 0.81 Orange Red-orange... 5a,0-anhydrotetracycline.

E 0.95 Yellow- Orange 5a,6-anhydro-4- orange. dedimethylaminotetracycline.

0.38 Orange Green 4-dedimcthy1-amino4- hydroxy-tetracycline.

0.38 Red Red 4-dedimethylarnino'4- keto-4a,12a-deliydr0- 12,l2a-seeotetraeycline- 12-oic acid 6,12-lactono.

0. 22 (Absorbing) 4-dedimethylamin0- 9-hydroxytetracyeline.

0.57 Orange Green I 0.57 Yellow"- Orange.

0.12 Orange--. I

0.02 Dull rcdt orange. orange.

(Absorbing) r lined, the chromatogram is then exposed momentarily to ammonia vapor and re-examined under ultraviolet light. Compounds with antibacterial activity are detected by bioautography in the usual manner using an overnight culture of Staphylococcus aureus strain 209P as test organism. The reaction products and some of their properties are listed in the following table. Under Inhibitory Activity a plus sign indicates the compound possesses antibacetrial activity and a minus sign indicates the compound does not possess antibacetrial activity against the test organism named above.

Compound B is isolated in pure crystalline form by preparative paper chromatography, subsequent countercurrent distribution in hexane ethylacetate-methanolwater (4:4:3z4) (14:10), and crystallization from ethanol-water.

Properties of Compound B [111 2 60 (c. 0.6, methanol).

Elemental analysis: Anhydrous (calcd. from hydrate): C, 59.69; H, 4.66; N, 3.39; C H O N: C, 59.85; H, 4.77; N, 3.50.

IR (Nujol): 5.65 ('y lactone), sh 6.03, 6.15, 6.36, 6.90 (6.90 band also present in CHCI spectrum).

UV spectra (95% ethanol 0.1 -N HCl): 217 mu (e=16,600), 270 (21,700), 345 (4,600).

(Ethanolic 0.1 N NaOH): 245 (19,700), 273 (15,900), 382 (6,800).

(Above after standing 0.5-2 hrs.): 246 (22,900), 274 (18,700), 341 (7,600).

(Above after acidification): 211 (20,900), 269 (19,- 800).

EXAMPLE 2 The reaction is carried out as in Example 1 with the exception that the original reaction mixture has been modified to have the following composition:

Volume Final (mL) Component concentration 1.0 Potassium citrate buffer, pH 5.0 0.1 M 1.0 Tetracycline hydrochloride 500 gJml. 1.0 Horseradish peroxidase (Worthington, 50 rig/m1.

Grade D). 1.0 Potassium or ammonium formate 5.0 mgJml. 1.0 Dihydroxyfumaric acid (adjusted to pH 10 mgJml.

5.0 with potassium hydroxide).

The result of this modification is an alternation in the number and relative concentration of the end products which accumulate. These are shown in the following table, wherein plus signs indicate an increase in quantity of the compound in question and a minus sign indicates no increase in quantity relative to Example 1 (the same letters are used to indicate the compound as were used in the preceding table):

6 EXAMPLE 3 The reaction is carried out as in Example 1 with the exception that the original reaction mixture has been modified to have the following composition:

Volume Final (ml) Component concentration 1.0 Potassium acetate bufier, pH 4.5 0.1 M

1.0 Tetracycline hydrochloride 500 gJml.

1.0 Horseradish peroxidase (Worthington, 50 ug./ ml.

Grade D).

1.0 Chloro-p-phenylenediamine dihydropgJml.

chloride.

1.0 Dihydroxyfumarie acid (adjusted to pH 10 mgJml.

4.5 with potassium hydroxide).

The result of this modification is an alteration of the number and relative concentration of the end products which accumulate as compared to Example 1. These are shown in the following table:

TABLE Stimulation due to chloro-p-phenylenediamine Product: dihydrochloride A +++-l- B C G H I J K EXAMPLE 4 The reaction is carried out as in Example 3 but substituting 1.0 m1. of potassium formate, at a final concentration of 0.1 M, for the potassium acetate buffer. This buffers the reaction medium at pH 4.0. The result of this modification as compared to Example 1 is shown in the following table:

The reaction is carried out as described in Example 2 except that a crude press juice of fresh horseradish root is substituted for the partially purified peroxidase. Essentially the same results are obtained as were obtained in Example 2.

7 EXAMPLE 6 The reaction is carried out as described in Example 1 with the exception that a highly purified preparation of horseradish peroxidase is employed (Worthington Biochemical Corporation, Grade A). Essentially the same results are obtained as were obtained in Example 1.

EXAMPLE 7 To each of forty 500 ml. Erlenmeyer flasks is added 100 ml. of the following reaction mixture:

Final concentration Volume 20 0.5 M potassium formats butter pH 4.0-... 0.1 M

Distilled water Component 4 with KOH). 10 Horseradish peroxidase (Worthington,

Grade D 50 ugJml.

The flasks are shaken on a rotary shaker at 25 for 60 minutes, when the reaction is essentially complete. The major products formed are 4-hydroxy-4-dedimethylaminotetracycline and 4-dedimethylamino-4-keto-4a,12adehydro-12,12a-secotetracycline-12-oic acid 6,12-lactone. These are separated from each other and from unreacted substrate tetracycline in the manner described below.

The contents of the flasks are pooled and extracted with 500, 250 and 250 ml. portions of ethyl acetate. The ethyl acetate extracts are combined, dried with anhydrous sodium sulfate, filtered, then evaporated to dryness at a temperature less than 35 under reduced pressure. The dry weight of the orange-brown solid thus obtained is about 1.15 grams.

This material is further purified by column chromatography as follows: 500 g. Whatman Standard Grade Cellulose Powder is slurried with Mcllvaines buffer, pH 4.6, filtered and dried. The dried, buffered powder is bydrated by wetting with 80% aqueous acetone and then aerating to remove the acetone. The moist powder is then suspended in n-hexanezethyl acetate (2:1) and poured as a column 6.7 x 41 cm. The orange-brown solid obtained above (about 1.15 g.) is dissolved in hexanezethyl acetate (2:1) and loaded onto the column, and elution with the same solvent is begun at the rate of 0.5 mL/min. with fractions collected every 20 minutes. Each fraction is analyzed chromatographicall using Whatman No. 1 paper buffered at pH 4.6 with McIlvaines buffer, developed with hexanezethyl acetate (1:1). Fractions containing the products free of the unreacted substrate tetracycline are pooled, dried with anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure at 35 About 258 mg. of a yellow powder is obtained. This yellow powder cannot be further purified in a satisfactory manner by chromatography, but is resolved into its two components by the following procedure.

The yellow powder is triturated with methanol to give a methanol-soluble and a methanol-insoluble fraction. The methanol-insoluble portion (41 mg.) proves to be pure 4-dedimethylamino 4 keto 4a,l2a dehydro- 12,12a-secotetracycline-12-oic acid 6,12-lactone, which possesses a mustard-yellow color. The methanol-soluble material is chromatographed on Whatman No. 1 paper buffered at pH 4.6 with hexanezethyl acetate as above, the ultraviolet absorbing bands at R; ca. 0.38 are cut out and eluted with methanol. The solution is taken to dryness, and the residue is then partitioned between saturated ammonium sulfate solution and ethyl acetate. The ethyl acetate extract is dried, filtered, and taken to dryness as above. The residue is dissolved in a small volume of ethyl acetate and upon the addition of a large excess of hexane a white, amorphous powder precipitates. This product (14 mg. yield) is essentially pure 4-hydroxy-4- dedimethylaminotetracycline.

4-dedimethylamino- 4-keto-4a,12a-dehydro- 12,12a-secotetracyeline- 12-010 acid 6,12-lactone 4-hydroxy-4-dedimethylaminotetracycline Physical appearance... White amorphous Mustard -yellow powder. pow er. M.P 225.5227.5 decomp. 61? ws "antes-2255a; '2;5 i;ii 2'09(459) s ectra 1 c a 365 300). sh 331 (116), 422 12 (0.1 N HCl) Same as above (stable sh 244 (263), 274 (670),

over 20 hr. period). 295 (400), 338 (124). (0.1 N NaOII) 243 (504), 270 (405),

384 (356) (on standing over 1 hr. decomposed to products having nondescript UV B 11 1i rig t? 31 as 6 IR K r s i0 -Z. 2.95, 3.05, 5.02, 6.08,

EXAMPLE 8 The reaction is carried out essentially as in Example 1 with the exception that a crude peroxidase preparation from the microorganism Myrothecium verrucaria ATCC 9095 (American Type Culture Collection, Washington, DC.) is employed and the incubation temperature is 25. The peroxidase is obtained by growing the microorganism for five days at 25 in a soybean meal-glucose medium of the following compositions: G Archer-Daniels-Midland extracted soybean meal 30 Glucose 50 CaCO, 7 Distilled water, to make 1 liter.

The mycelium is recovered by filtration, dried with anhydrous acetone in the usual manner, and ground to a fine powder. The dry powder is stored in a desiccator at about 4 until immediately before use, when it is extracted with distilled water. The solution is used as the source of the enzyme. Essentially the same results are obtained as were obtained in Example 1.

EXAMPLE 9 The reaction is carried out as in Example 8 except that the solution of the enzyme is partially purified by precipitation with ammonium sulfate. Powdered ammonium sulfate is added to the distilled water extract of the acetone powder of M. verrucaria to the point of 50% saturation. The protein precipitate which forms is collected by centrifugation, and is redissolved in water. This concentrated solution of protein can be used as such, or freed of residual ammonium sulfate by dialysis against water overnight at 4-6. Essentially the same results are obtained as were obtained in Example 1.

EXAMPLE 10 The compound 4-dedimethylamino-4-keto-4a,12a-dehydro-l2,12a-secotetracycline-12-oic 6,12-lactone can also be prepared by a nonenzymatic procedure as follows. Tetracycline is treated with m-chlorperbenzoic acid in dioxane at room temperature. The reaction is followed by quenching with potassium iodide solution and titration of liberated iodine with standard thiosulfate solution. It is found that 1 mole of the peracid is consumed very rapidly (12 sec. at 24.5") and the uptake of peracid continues rapidly, gradually tapering off at 2.5 moles (90 sec). Paper chromatographic study of the reaction over a range of peracid concentrations shows the optimum concentrations to be 1.5 moles of peracid to 1 mole of tetracycline. For preparative purposes 1.6 moles m-chlorperbenzoic acid is reacted with 1 mole tetracycline in methanol. The mixture is allowed to stand at 24.526 until a starch-iodide test shows all the peracid to be consumed. The .methanol is removed and the red, oily residue is extracted with several portions of boiling hexane. The residue is triturated with ethyl acetate to produce a brickred powder. Isolation of the product through preparative paper chromatography and comparison with data of the product produced enzymatically shows them to be identical and establishes the identity as that of the compound named above.

10 EXAMPLE 13 The reaction is carried out as in Example 1 except that S-hydroxytetracycline is used in place of tetracycline as the substrate. The reaction products and some of their 5 properties are listed in the following table. Under Inhibi. EXAMPLE 11 tory Activity a plus sign indicates antibacterial ac- The new compound 4-hydroxy-4-dedimethylaminotettivity against Staphylococcus aureus strain 209-P, and a racycline is also prepared nonenzymatically from tetracyminus sign indicates that the compound does not cline in the following way: possess antibacterial activity against this strain.

I TABLE Inhibitory Fluorescence Fluorescence Product Rt activity before NH; after NH; Identity P 0.72 Orange Green 4-dediniethylamino-5- hydroxytetracycline. Q 0.56 ...d Orange 4-dedimethylamine5- hydroxy-12,12asecotetracyeliue-iZ-oic acid 6,12-1actoue. 0. 90 r z 0177 r 0. 32

Tetracycline hydrochloride (0.5-5.0 mg./ml., optimum EXAMPLE 14 c9ncemrat1n ls giifg i 5 The reaction is carried out as in Example 1 except that m formate ut: PH 6% foxy if is 25 6-demethyltetracycline is used in place of tetracycline as adqed as f potassmm sat 0 substrate. The reaction products and some of their propconcentfatlflfl 10 the macho mmaied erties are listed in the following table. Under Inhibitory y the addltloll a dlhlte Solution f hydrogen peroxfde Activity a plus sign indicates antibacterial activity (fin al concentration O.03-3.0%, 1 f f lfi against S. aureus 209-P, and a minus sign indicates The reaction miXfllTe 1S Shaken 111 at that the compound does not possess antibacterial activity until all traces of the dihydroxyfumaric acid suspension against this strain.

TABLE Inhlbitory Fluorescence Fluorescence Product R! activity before NH after NHa Identity 0.94 Brown Brown 4-dedimethylamino- 5a,6-anhydro-6- demethyltetracycllne. 0.89 Dull rose Dull rose 0.83 Yellow- Orange... 5a,6-anhydro-6- orange. demethyltetracycline. 0.77 Yeliow-green.. Green.. 4-dedimethylamino-6- demethyltetracycline. 0.70 Pink Pink 4 dedirnethylamino-6 demethyl-12,12aseco-tetracycline-12- 0 65 01c acid 6,12-lactone. 015s r 0. 51 4-hydroxy-4- surna s 0.39 Pink Pink r em y 0. (Absorbing) 0.31 Rose Rose 0. 27 (Absorbing) 4-dedimethylamino-6 demethyl-Q-hydroxyo 18 Wh Pink tetracycline. 0: 12 "'(Ilis'rbin i 0.09 R0 e Rose I disappear and the medium clears (ca. 2 hrs). The reac- What is claimed is: tion products are then extracted into ethyl acetate, and 1. A process for preparing tetracyclines which comare characterized chromatographically as described preprises subjecting a compound selected from the group viofisly. The above-named compound is found to be the consistlng of tetracycline, 6-demethyltetracycline and 5- major product, and comparison of its physical properties hydroxytetracycline, under aerobic conditions to the acdetrmined by analysis with those of the compound protlon of peroxidase in the presence of dihydroxyfumaric duced enzymatically proves its identity. acid.

2 A process for preparing 4-hydroxy-4-dedimethyl- EXAMPLE 12 aminotetracyclme, which comprises subjecting tetracycline under aerobic conditions to the action of peroxidase 4-hydroxy-4-dedimethylaminotetracycline 1n the presence of dihydroxyfumaric acid at a tempera- 4,12a-diacetate ture in the range of about 15 C. to about 37 C.

3. A compound of the formula A 0.10 g. sample of 4-hydroxy-4-dedimethylaminotet- OR OR racycline is treated with 1 ml. of acetic anhydride. After I 14 days at a temperature of 2530 the solution is taken 03 to dryness in vacuo at a temperature less than 35 The last trace of acetic anhydride is destroyed by the addition i; of water in the usual way, and the product is filtered, I washed with water, and dried. OH 0 H 0 Similarly, if other acid anhydrides are substituted for 0 acetic anhydride, other 4,12a-diesters are formed. R

wherein each R is selected from the group consisting of hydrogen and the acyl radical of a hydrocarbon carboxylic acid of less than twelve carbon atoms, and R' is selected from the group consisting of hydrogen and methyl.

4. 4-hydroxy-4-dedimethylaminotetracycline.

5. 4 hydroxy 4 dedimethylamino 6 demethyltetracycline.

6. The compound of claim 3, wherein both Rs are acetyl and R' is methyl.

7. A process for preparing 4-hydroxy-4-dedimethylaminotetracyclinc which comprises treating tetracycline with dihydroxyfumaric acid and hydrogen peroxide.

References Cited UNITED STATES PATENTS 3,375,276 3/1968 Neidleman et a1.

3,196,164 7/1965 Lucas et a1. 260-3433 5 3,198,810 8/1965 Wygrant et al. 260-3433 2,812,349 11/1957 Gordon 260-490 3,021,360 2/1962 Pohland 260-590 ALEX MAZEL, Primary Examiner ANNE MARIE T. TIGHE, Assistant Examiner US. Cl. X.R.

Notice of Adverse Decision in Interference In Interference No. 98,970, involving Patent No. 3,462,487, R. W. Kinney, S. L. Neidlenmn, F. L. Weisenborn and J. S. P. Schwarz, CHEMICAL COM- POUNDS AND METHODS FOR PREPARING THE SAME, final judgment adverse to the patentees was rendered Oct. 12, 1976, as to claim 4.

[Oflicz'al Gazette February 1, 1.977.] 

